Penicillin salts of anion exchange resins



Patented Sept. 14, 1954 STATES ATNT OFFICE PENICILLIN SALTS F ANIONEXCHANGE RESINS No Drawing. Application April 25, 1952, Serial No.284,450

6 Claims.

anion-exchange resins. It deals also with a method for isolating,purifying or recovering penicillin.

With greater particularity this invention concerns on the one handquaternary ammonium anion-exchange resins which hold on the functionalgroups thereof penicillinate ions, these being penicillin salts of theseanion-exchange resins, and on the other hand it also concerns penicillinsalts formed from penicillinic acid and anion-exchange resins havingamino groups as the functional groups thereof, these resins havingprimary, secondary and/or tertiary amino substituents as the functionalgroups thereof.

Further, this invention relates to processes whereby the above salts areformed. With quaternary ammonium anion-exchange resins a solutioncontaining penicillin is brought in contact with a said resin and theoriginal anion of the resin is replaced by the penicillinate ion to forma resin salt. In one important aspect of this invention solutionscontaining penicillin are conditioned by bringing them in contact with arelatively dense quaternary ammonium anionexchange resin in the form ofa salt in which the anion is other than penicillinate and then bringingthe conditioned solution in contact with an adsorbent form of aquaternary ammonium anion-exchange resin in salt form, a porous resinbased on a copolymer from 99.5 to 98 parts by weight of styrene and 0.5to 2 parts of (polyvinyl) benzene being particularly useful. The saltsformed from quaternary ammonium anion-exchange resins may be representedas to the func- R, R", R representing the quaternary-formingN-substituent and Penthe penicillinate ion.

The invention also deals with separating penicillinic acid and reactingit with a basic anionexchange resin to form a salt. The aminocontaininganion-exchange resins are the exchangers best adapted to be used in thisway. They can be considered to form salts by addition of an acidmoleculeto the amino group. The sal thus formed can be formulated as --XN(R)(R").H pen; where X represents the resin structure, R and R" representhydrogen or other amine-forming substituents, and H pen representspenicillinic acid.

The present application is a continuation-inpart of my applicationSerial No. 96,133, filed on May 28, 1949, now abandoned, which in turnis a continuation-in-part of my application Serial No. 759,308, filed onJuly 5, 1947, now U. S. Patent No. 2,591,573. This patent describes thepreparation of quaternary ammonium anion-exchange resins based oncross-linked styrene polymers. The aromatic rings of these polymerscarry quaternary ammonium groups attached thereto through alkylenegroups, usually methylene groups.

In application Serial No. 96,133, filed May 29, 1949, I have described aconvenient process for isolating or purifying penicillin with the aid ofquaternary ammonium anion-exchange resins. I have also there shown thatthese resins combine with the anion of penicillin, thereby forming aninsoluble penicillin salt. Under proper conditions, as in the presenceof a solution containing an electrolyte, the penicillin anion isrelatively rapidly and readily released from the anion exchanger; theanion in preponderance replacing the anion of penicillin. The penicillinsalt of an anion-exchange resin thus offers a means of collecting andpurifying penicillin. It also becomes a source for supplying penicillinto environments in which anions from inorganic salts and the like arepresent for exchange. The penicillin salts of anion-exchange resins areparticularly useful as supplements for animal and poultry feeds tosupply penicillin.

Penicillin salts of quaternary ammonium anion-exchange resins areprepared by bringing together an aqueous solution of penicillin and aquaternary ammonium anion-exchange resin having an exchangeable anionother than that of penicillin. This anion is commonly one such aschloride, phosphate, or sulfate or one such as bicarbonate or acetate.The operation is most conveniently carried out by flowing a solution ofpenicillin through a bed containing the anionexchange resin. Thesolution may contain from a trace of penicillin up to saturation.

Alternatively, an aqueous solution containing penicillin is treated witha quaternary ammonium anion-exchange resin. The solution may be treatedwith several batches of such resin to ensure practically completeremoval or" penicillin from solution. Subsequently used batches of resinmay be used for treating a fresh solution of penicillin to increase theamount of penicillin taken up by the reactive centers of the resin. Itis not, however, essential that every quaternary ammonium salt group beconverted to a penicillinate group.

After the penicillin salt of an anion-exchange resin has been formed,treating solution may be rinsed therefrom. The resin may then be driedat low to moderate temperatures, desirably under reduced pressure, if itis desired that the penicillin be used in this form. If the penicillinsalt is used primarily as a collecting agent, it may be freed oftreating solution and the penicillinate ion eluted therefrom, as with anaqueous 5% to solution of sodium chloride or a buffer solution.

The exchange step and the elution step are best performed attemperatures between 0 and C. The conditions of pH of the solutions areusually held between values of 4.5 and 8.5. If lower pHs are used, thenthe temperature should be held low, a range of 0 to 10C. being thengenerally practical. Drying is usually accomplished between 0 and C.,best under reduced pressure. Freeze drying may desirable be applied toremove water from the penicillin-resin salt.

The quaternary ammonium anion-exchange resins generally used in theabove-described procedure are insoluble resinous compositions formed bychloromethylating a styrene-(polyvinyl)benzene copolymer containing apredominant amount of styrene and reacting the chloromethylated productwith a tertiary amine. The amine is preferably one of small molecularsize, such as trimethylamine, triethylamine, dimethylhydroxyethylamine,benzyldimethylamine, ethyldimethylamine, cyclohexyldimethylamine, etc.,the exact nature of the nitrogen substituents not being a critical ordetermining factor here.

Although resins containing relatively few quaternary ammonium groupswill take up penicillin at these points, it is desirable from apractical point of view to have sufiicient quaternary ammonium groups onthe resin nuclei to give good capacity to the resin. In generalcapacities are very satisfactory when two to six quaternary ammoniumgroups are present for every four aromatic rings, although resins arestill useful with as little as one such group per 15 aromatichydrocarbon nuclei in the copolymer. The maximum number of such groupsoccurs, of course, when every available position in the copolymer ischloromethylated and converted to the quaternary ammonium form byreaction with a tertiary amine.

A factor which is of interest in the formation and use of penicillinsalts of the anion-exchange resins is the physical nature of theseresins. When the resins are so formed as to be quite porous, they can bereadily and substantially completely converted to the penicillin saltform. The penicillinate ion can apparently enter all of the pores ofsuch resins readily and in a short time and replace the salt-forminganions first present at the site of the active groupings. With lessporous resins the exchange is less readily effected and often lesscompletely, unless the less porous resins are broken up into fineparticles so that they present large surfaces upon which the exchange ofanions takes place rapidly.

As I have explained in application Serial No. 96,133, copolymers ofstyrene made with 0.5% to 2% of divinylbenzene give finishedion-exchange resins of low density and high porosity. As cross-linkingincreases, either through use of higher percentages of a cross-linkingagent such as divinylbenzene or other polyvinyl compound, the finishedion-exchange resin becomes more dense and the pores thereof becomeeither smaller or fewer. Cross-linkages established through methylenegroups from the halomethylating agent are somewhat intermediate ineffect between the above pcrous resins and less porous forms nowdescribed. Copolymers from styrene copolymers produced with 4% to 8% ormore of divinylbenzene are definitely more dense than those made with0.5% to 2% of divinylbenzene. In granular form they take up lesspenicillin. Yet the available groups of these denser resins definitelyact as centers where the penicillinate ion is exchanged for other anion.The proportion of penicillin which the denser resins take up is greatlyincreased as the particle size is decreased. Thus finely powdered resinsof this type can be used to form penicillin salts by the batch methoddescribed above and these can hold a good proportion of enicillin on aweight basis.

A type of quaternary ammonium anion-exchange resins which are effectivefor exchanging anions such as chloride and the like with thepenicillinate ion and which have a favorable and high capacity for suchan ion is formed by proliferous polymerization as described by G. W.Bodamer in application Serial No. 226,094, filed May 12, 1951, now U. S.Patent No. 2,597,440, in the hands of a common assignee. These exchangeresins are equivalent to the porous resins derived from copolymers basedon styrene and divinylbenzene in weight ratios from 99.5 to 98 parts ofthe former to 0.5 to 2 parts of the latter.

The physical differences between quaternary ammonium anion-exchangeresins made in a porous form, as with 0.5% to 2% of divinylbenzene and99.5 to 98 of styrene, and less porous resins, such as those made with4% to 8% or more of divinylbenzene, make possible an interesting anduseful process for assisting in the recovery or preparation andisolation of penicillin.

It has been found that of the various inorganic anions and the likewhich are ordinarily encountered the chloride ion has the least effectupon adsorption of penicillin from its solutions. For this reason it isdesirable to pass a penicillin solution containing miscellaneous ionsover a relatively dense form of the anion-exchange resin in the chlorideform and best in a size suitable for use in columns. Since the denseforms exchange all the usual anions and have on a volume basis somewhatbetter capacity than highly porous forms, passage over the dense formconditions the solution. The solution is then contacted with a porousquaternary ammonium anion-exchange resin. Alternatively, it is contactedwith a finely divided anion-exchange resin which may be prepared, ifdesired, from a relatively dense form, best in the batch process.

For example, a solution containing 3.56 grams of sodium penicillin G perliter is passed at about 20 C. through a column containing a granularquaternary ammonium anion-exchange resin in its chloride form, thisresin being derived from a copolymer of 88.77% of styrene and 11.23% ofcommercial divinylbenzene, of which 53.4% is in fact divinylbenzene and46.6% is ethylvinylbenzene, through chloromethylation and reaction withtrimethylamine. This resin contains 4.2% of nitrogen by analysis and hasa capacity of 3.24 milliequivalents per gram or 1.29 milliequivalentsper milliliter. In the passage of the solution through the column thisresin takes up 0.012 milliequivalent per milliliter of penicillinateion. The effluent from the column is passed through a column containinga quaternary ammonium anion-exchange resin similarly prepared but basedon a resin from 98.5% of styrene and 1.5%

of divinyl benzene. This resin has a capacity of about onemilliequivalent per milliliter. This resin takes up 036 milliequivalentof penicillin per milliliter, as determined by iodiometric titration.The anion-exchange resin salt thus prepared contains a high proportionof penicillin in a relatively pure form. After the salt has been dried,it is stable and useful as a source of penicillin.

A solution of 90' grams of potassium penicillinate in a liter of wateris passed at room temperature through a column of a porous quaternaryammonium anion-exchange resin based on a copolymer from styrene anddivinyl benzene in a 995:0.5 ratio, which has been chloromethylated andthen reacted with dimethylhydroxyethylamine to form quaternary ammoniumchloride groups. This resin takes up 0.7 gram of penicillin ion per gramof resin, thus forming the penicillin salt. The resin is rinsed. withdeionized water, sucked free of water on a filter pad, and dried underreduced pressure with warming to about 35 C. The roduct is stable whenstored.

A broth resulting from the culturing of Penicz'ZZinr-i notoiam isfiltered free of solid material. it is passed over a granular resin fromstyrene and divinylbenzene in a 92:8 weight ratio which beenchloroinethylated and reacted with benzyldirnethylamine. The efiiuentfrom this treatment is passed through a column packed with a -5'3 me 1resin obtained from a copolymer of styrene and divinylbenzene in a 99-:1 weight ratio, which has been chlorornethylated and reacted withtrirnethylamine. This resin takes up 0.65 grain of penicillin ion pergram of resin to form the penicillin salt thereof. The resin is rinsedwith deionized water and dried under reduced pressure. This product isstable and suitable for use in poultry or animal feeds.

tered broth is passed through another porof the same porous resin in acolumn. The n i" rinsed by upflow with deionized water.

solution of sodium chloride. The chloride the penicillin. ions. Theeffluent is ed to 0 C. and treated with cold 20% phosp ioric acid to apH of 2.5 and the acidified solution is extracted with cold butylacetate to remove penicillinic acid from the aqueous layer. The solventsolution is washed with water which is acidified to 3 with a littlephosphoric acid and the washed solution is treated with a soluon ofprocaine in butyl acetate. The penicillin "oc- .le salt precipitates andis readily separated 3y filtration.

A portion of the above cold butyl acetate solution of penicillin in itsacid form is contacted w h a finely divided quaternary ammonium oexchange resin prepared from a copolymer st no and divinyl. benzene in a95:5 weight ratio hich has been chloromethylated and reacted withdirnethyihydroxyethylamine to give the quaternary ammonium chlorideresin which has then been treated with 5% sodium bicarbonate solutionseveral times to yield the bicarbonate form of the exchanger. The resinand solution are separated on a filter. After the resin is sucked freeof solution, it is rinsed with a little butyl acetate and with water,and dried under reduced pressure. A stable penicllin-resin salt is thusobtained which contains about 0.7 gram of penicillin ion per gram ofresin.

Another portion of the cold acid extract in butyl acetate is passed veryslowly througha basic resin in a porous form, prepared from styrene andthat chill divinyl benzene in a 99:1 weight ratio, the copolymer ofwhich has been chloromethylated, treated with diethylenetriamine to formamine hydrochloride groups, and washed with a 5% sodium carbonatesolution to place the resin in its basic form. A flow rate of about 0.25to 0.5 gallon of solution per cubic foot of resin per minute is usedhere. The resin takes up the acid penicillin, thereby forming apenicillin salt with this anion-exchange resin. Solvent and resin saltare separated by filtration. The salt is rinsed with butyl acetate andwith water and then dried under reduced pressure. The resulting productis insoluble and stable in a dry form. It is not, however, apparently asstable as the penicillin salts of quaternary ammonium anion-exchangeresins and it releases penicillin more slowly than the latter resins.

In the removal of penicillinic acid from a solvent extract a veryadvantageous method is based on use of a powdered basic anion-exchangeresin of the type used just above. Solvent solution of acid and powderedresin are mixed and stirred for 12 to 24 hours. The anion-exchange saltis thus formed. The salt formed in either way is useful for feedsupplements.

Apparently penicillin is released from the above type of resin ratherslowly, but over a relatively long period of time. This is believedadvantageous for use of salts of this type as feed supplemerits.

Nitrogenous resins such as used above are available from the reaction ofchlorornethylated polymeric cross-linked styrene and primary orsecondary amines, as described in my U. S. Patent No. 2,591,574, whichissued on April 1, 1952. In-

stead of the resin from diethylenetriamine shown above, there may beused any of the other basic anion-exchange resins prepared by the methoddescribed in the aforesaid patent. The polyalkylenepolyamines provideone particularly interesting subclass of amino resins for forming saltswith penicillinic acids. Another subclass comprises basic resins formedby reacting the chloroznethylated copolymer with simple monoamines, suchas dimethylamine, methylamine, diethlyamine, ethylamine, benzylarnine,butylmethylamine or the like, and converting the re- Suitinghydrochloride resin to the basic form. If penicillin. salts of aminoresins are desired, it is preferred that the basic resin depend foractivity on a tertiary amino group, as is present in the resin formedfrom a chloromethylated crosslinked styrene polymer and a secondaryamine such as dimethylamine. These have a very favorable. behavior forreacting with penioillinic acid.

The amino anion-exchange resins prepared with primary or secondaryamines, like the quaternary resins prepared with tertiary amines, can bemade in different densities and porosities by varying proportions ofcross-linking agent conditions of reaction. The amino resins based on.copolymersv from styrene and (polyvinyhbenzene in ratios from 995:0.5 to97 :3 by weight are definitely to be preferred for this class ofexchangers...

In place of the just described amino anion exchange resins, there may beused other anionexchange resins, depending also on basic amino groupingsfor their activity and salt-forming capacity. There may be mentioned,although the properties thereof are not entirely favorable, theanion-exchange resins prepared from methylolforming phenol-s,formaldehyde, and ammonia or 7. reactive amines (i. e., those havinghydrogen on the nitrogen). These resins can be made in a number of ways,all of Which lead to essentially the same sort of resins having basicamine groups.

The resins of this type are sufiiciently described in issued patents.For example, U. S. 2,356,151 and 2,402,384 give an adequate descriptionof typical resins of this sort for present purposes and possiblevariations of these will be evident. Still other types of basicanion-exchange resins may be used to form salts with penicillinic acid.But these latter types of amino resins are not ordinarily of as greatimportance as the resins which are based on styrene copolymers such asdescribed above. This may be because these latter kinds of resins do nothave as suitable physical forms and also because there may be somedifference in the way the penicillin group is held.

There are some additional types of weakly basic anion-exchange resinswhich have useful properties for forming salts with penicillinic acid.One of these is described by G. W. Bodamer in application Serial No.226,093, filed May 21, 1951, now U. S. Patent No. 2,597,439,aminocontaining anion-exchange resins being formed by proliferouspolymerization. These present a very large surface in which the aminogroups are fully available for forming salts with a large ion such asthat from penicillin.

Another type of amino resin of value is that formed by ammonolysis ofcross-linked acrylate resins with amines such as polyalkylenepolyamines.For example, ethyl acrylate is copolymerized with divinylbenzene andthis copolymer is reacted with diethylenetriamine. Details of thepreparation of such resins are presented in application Serial No.282,275, filed April 14, 1952, by H. J. Schneider, and like theabove-noted patent applications, this application is in the hands of acommon assignee. These resins are of high capacity and are effective forforming penicillin salts with penicillinic acid.

There is a fundamental difference which should be mentioned between theclass of quaternary ammonium anion-exchange resins and the class ofamino resins. The former in their alkaline form exist as strong basescomparable to sodium or potassium hydroxides. The liydroxyl form ofthese resins should, therefore, be used with penicillins only withconsiderable caution. Yet because of the nature of these exchangerstheir free bases need not be used, for the anions of these resins can betruly exchanged for other anions, the penicillinate ion thus beingcapable of replacing anions such as chloride and or" being replaced. Ifa basic quaternary ammonium exchange resin should be desired, forinstance, so as to react with acid, the resin of choice to use is aquaternary resin in its bicarbonate form, for then the pI-Is encounteredwill not exceed about 8.5.

The anion-exchange resins depending for their activity upon amino groupsdo not have the above capability of free exchange of anions, but ratherdepend upon their basic character and must be used in their basic stateto form salts of enicillinic acids. These salts are prepared by bringingsolutions of the penicillinic acids essentially free of other acids intocontact with the basic resins. The salts thus formed can graduallysupply penicillin to aqueous environments in which they are placed.They, too, are suitable for supplements to animal and poultry feeds andfor other uses.

A potential use of quaternary ammonium anion-exchange resins is in therecovery of penicillin from weak broths or offgrade batches or fromtailings from conventional processing steps. The broths or liquors aredesirably conditioned by being passed over a dense, granular quaternaryammonium exchange resin in its chloride form and then over a porousresin as shown above. Penicillin retained by the dense resin can beeluted as with a sodium chloride solution and the eluted resin reused.Alternatively, the resin can be reduced to a powder form, saturated withmore penicillin, and then used as the penicillin salt.

The quaternary ammonium anion-exchange resins in a porous form makepossible another important improvement in the process of preparingpenicillin. Penicillin is elaborated by culturing Penicillium notatum orchrosogenum in a nutrient medium. As penicillin is developed, the brothis passed by upfiow through a bed of a quaternary ammoniumanion-exchange resin in the form of an inorganic salt or of a salt withan acid such as acetic. A constant side stream may, for example, bepumped from the culture up through the bed. The effluent from the bed isreturned to the culture tank. In order to permit free passage of culturemedium through the bed, the bed is expanded 50% to 75% by the flow ofbroth. This method has the advantage that the level of penicillin in thecultured broth is not permitted to become high, penicillin is removedbefore penicillin has long been in contact with penicillinase, and adifficult filtration problem is avoided. When the resin is sufiicientlysaturated with penicillin, it is washed, preferably by upfiow to removematerial not previously floated off. The bed is then eluted toconcentrate penicillin in the effluent, as has been described, andWorked up by conventional steps or both other methods, such as describedabove. Alternatively, the anionexchange resin-penicillin salt may beused as such.

As nearly as can be determined all of the types of penicillin are takenup by the anion-exchange resins and while most emphasis is usuallyplaced on penicillin G because of its marked antibiotic activity, itshould be noted that other forms of penicillin, including penicillins F,K, or X, form salts with anion-exchange resins and that mixtures of thevarious types of penicillins can be used quite as well as the singletypes.

I claim:

1. A process for preparing a penicillin salt of a quaternary ammoniumanion-exchange resin which comprises passing at a temperature from 0 to30 C. an aqueous solution containing penicillin and having a pH of 4.5to 8.5 over a relatively dense anion-exchange quaternary ammonium resinin its chloride form and then contacting the solution with a porousquaternary ammonium anion-exchange resin in the form of its chloride,said porous resin being a copolymer of 99.5 to 98 parts by weight ofstyrene and 0.5 to 2 parts by weight of divinylbenzene having itsquaternary ammonium chloride groups attached to aromatic nuclei throughmethylene linkages, said quaternary ammonium chloride groups havingthree methyl groups as N-substituents.

2. A process for preparing a penicillin salt of a quaternary ammoniumanion-exchange resin which comprises passing at a temperature of 0 to 30C. an aqueous solution containing penicillin and having a pH of 4.5 to8.5 over a relative- 1y dense anion-exchange quaternary ammonium resinin its chloride form and then contacting the solution with a porousquaternary ammonium anion-exchange resin in the form of its chloride,said porous resin being a copolymer of 99.5 to 98 parts by weight ofstyrene and 0.5 to 2 parts by weight of divinylbenzene having itsquaternary ammonium chloride groups attached to aromatic nuclei throughmethylene linkages, said quaternary ammonium chloride groups having twomethyl groups and one hydroxyethyl group as N-substituents.

3. A process for preparing a penicillin salt of a quaternary ammoniumanion-exchange resin which comprises passing at a temperature of to 30C. an aqueous solution containing penicillin and having a pH of 4.5 to8.5 over a relatively dense anion-exchange quaternary ammonium resin inits chloride form and then contacting the solution with a porousquaternary ammonium anion-exchange resin in the form of its chloride,said porous resin being a copolymer of 99.5 to 98 parts by weight ofstyrene and 0.5 to 2 parts by weight of divinylbenzene having itsquaternary ammonium chloride groups attached to aromatic nuclei throughmethylene linkages, said quaternary ammonium chloride groups having asthe other N-substituents at least on member of the class consisting ofmethyl, ethyl, hydroxethyl, and benzyl groups.

4. A penicillin salt of an anion-exchange resin of a copolymer of 99.5to 98 parts by weight of styrene and 0.5 to 2 parts by weight ofdivinylbenzene, said copolymer having quaternary ammonium groupsattached to the aromatic nuclei thereof through the methylene linkage,having as N-substituents at least one member from the class consistingof methyl, ethyl, hydroxyethyl, and benzyl groups and havingpenicillinate ions as anions thereof.

5. A penicillin salt of an anion-exchange resin of a copolymer of 99.5to 98 parts by weight of styrene and 0.5 to 2 parts of divinylbenzene,said copolymer having quaternary ammonium groups attached to thearomatic nuclei thereof through the methylene linkage, 'having threemethyl groups as N-substituents, and having penicillinate ions as anionsthereof.

6. A penicillin salt of an anion-exchange resin of a copolymer of 99.5to 98 parts by weight of styrene and 0.5 to 2 parts of divinylbenzene,said copolymer having quaternary ammonium groups attached to thearomatic nuclei thereof through the methylene linkage, having two methylgroups and one hydroxyethyl group as N- substituents, and havingpenicillinate ions as anions thereof.

References Cited in the file of this patent UNITED STATES PATENTS NumberName Date 2,479,832 Grant Aug. 23, 1949 2,629,710 McBurney Feb. 24, 19532,656,298 Loewe Oct. 20, 1953 OTHER REFERENCES Cruz-Coke et aL:Science," vol. 101 (1945), p. 340.

Penn. State Report (WPB Contract WPB-126) 44-76, August 24, 1944, 16 pp.

Penn. State Report (WPB Contract WPB-126) 44-93, October 3, 1944, 9 pp.

Penn. State Report (WPB Contract WPB-126), 44-100, October 19, 1944, 12pp.

Penn State Report (WPB Contract WPB-126), 44-107, November 16, 1944, 7pp.

Penn. State Report (WPB Contract WPB-126) 44-108, November 17, 1944, 9pp.

4. A PENICILLIN SALT OF AN ANION-EXCHANGE RESIN OF A COPOLYMER OF 99.5TO 98 PARTS BY WEIGHT OF STYRENE AND 0.5 TO 2 PARTS BY WEIGHT OFDIVINYLBENZENE, SAID COPOLYMER HAVING QUATERNARY AMMONIUM GROUP ATTACHEDTO THE AROMATIC NUCLEI THEREOF THROUGH THE METHYLENE LINKAGE, HAVING ASN-SUBSTITUENTS AT LEAST ONE MEMBER FROM THE CLASS CONSISTING OF METHYL,ETHYL, HYDROXYETHYL, AND BENZYL GROUPS AND HAVING PENICILLINATE IONS ASANIONS THEREOF.